Molecular targets for healing or treating wounds

ABSTRACT

The invention relates to at least one molecular target for healing or treating wounds and, in particular chronic, human wounds. The molecular target is PTPRK, or a protein 50% homologous therewith, and which retains the same activity as PTPRK protein. Further, the invention concerns a novel therapeutic for treating said wounds and a novel gene therapy approach, involving said molecular target, for treating said wounds.

FIELD OF THE INVENTION

The present invention relates to at least one molecular target forhealing or treating wounds and, in particular, human wounds. Moreparticularly still, the molecular target has application in thetreatment of chronic wounds. Further, the invention concerns a noveltherapeutic for treating said wounds and a novel gene therapy approach,involving said molecular target, for treating said wounds. Additionally,the invention concerns a method for treating wounds using saidtherapeutic or said gene therapy.

BACKGROUND OF THE INVENTION

In one form or another, chronic and poorly healing wounds constitute amajor burden on the UK health system. Moreover, in certain membercountries of the EU health expenses relating to wound healing arealready approaching the third most expensive drain on health carefunding.

A Chronic wound is herein defined as one exhibiting delayed or defectivehealing which does not progress through the predictable stages of thehealing process (as described below). Commonly, chronic wounds areclassified into three broad categories: venous ulcers, diabetic, andpressure ulcers. Long-term venous insufficiency accounts for 70% to 90%of chronic wounds and commonly affects the elderly. Venous insufficiencyresults in venous hypertension, in which blood flow is abrogatedresulting in subsequent ischaemia. Venous insufficiency can occur as aresult of obstructions to venous outflow or reflux due to valve damage.Following a period of ischaemia, tissue reperfusion can result inreperfusion injury, causing the tissue damage that leads to woundformation.

Chronic foot ulcers are a major complication of diabetes, accounting forup to 25% of all hospital admissions involving diabetes, and at a costto the UK National Health Service of £250M annually. Chronic foot ulcerscause substantial morbidity, impair the quality of life, and are themajor cause of lower limb amputation. Despite careful attention to footcare, as many as 25% of diabetics develop foot ulcers in theirlifetimes. The causes of lower limb ulceration are the same in diabeticsas in non-diabetics, namely neuropathy, ischaemia and trauma. However,this “pathogenic triad” predisposes wounds to infection, which can alsocontribute to the non-healing nature of the wounds.

Pressure wounds are another major resource health cost. They aretypically caused by failure to provide routine nursing or medical care.In the UK 412,000 people are affected annually by this sort of wound ata cost of £1.4-2.1 billion.

Furthermore, chronic wounds can also be categorised by whether they arecaused by surgery, burns, dermatitis, vasculitis or radiation.

Current wound treatment strategies involve removing pressure from thearea, debridement, wound dressing and management of infection: surgicalresection and vascular reconstruction may be required in more advanceddisease, which ultimately may necessitate amputation. These stragegiescommonly seek to address problems that are associated with chronicwounds, such as bacterial load, ischaemia, and imbalance of proteases,all of which can further affect the wound healing process.

The healing of a wound is controlled by complex biological processesthat involve a diverse number of cell types; complex interactionsbetween cells and tissues; the activation of the immune system and theactivation of the angiogenic process. Moreover, all of these processesinvolve a large number of molecules.

A typical healing process can be divided into 5 distinct, but closelyrelated, stages: clotting stage, acute inflammation stage, matrixdeposition stage, capillary formation stage and re-epithelialisationstage. A diverse number of factors are involved in and control each ofthese stages. Deficiencies in any aspect of the process may result indefective wound healing. Thus, a ‘normal’ healing process may bedefective as a result of either intrinsic or external factors, whichmanifest as ‘abnormal non-healing’ or ‘chronic’ wounds. It is thesechronic or ‘non-healing’ wounds that present the greatest challenge tothe quality of a patient's life and mounting expenses to the healthcaresystem.

A chronic wound often arises from failure to progress through the normalstages of wound healing, whereby an initial injury resulting in a woundcannot subsequently be repaired. Changes occur within the molecularenvironment of a chronic wound, such as high levels of inflammatorycytokines or proteases, and low levels of growth factors, these changesdetain or terminate the healing process and increase the potential forseptic infections. By enhancing or manipulating factors that contributeto wound healing it may therefore be possible to correct the process,thereby reducing the likely occurrence of a chronic wound, or accelerateits subsequent repair.

PTPRK

Protein tyrosine phosphatise receptor type K, PTPRK, is also known asDKFZp686C2268, DKFZp779N1045 and R-PTP-kappa. It is a member of theprotein tyrosine phosphatase (PTP) family. PTPs are known to besignalling molecules that regulate a variety of cellular processesincluding cell growth, differentiation, mitotic cycle, and oncogenictransformation. PTPRK possesses an extracellular region, a singletransmembrane region, and two tandem catalytic domains, and thusrepresents a receptor-type PTP. The extracellular region contains ameprin-A5 antigen-PTP mu (MAM) domain, an Ig-like domain and fourfibronectin type III-like repeats. Moreover, PTPRK has been shown tomediate homophilic intercellular interaction, possibly through theinteraction with beta- and gamma-catenin at adherens junctions.Expression of the PTPRK gene has been found to be stimulated by TGF-beta1, which may be important for the inhibition of keratinocyteproliferation. In cancer, PTPRK has been found to be suppressed inaggressive tumours as shown by our recent study in breast cancer (Sun etal, SABCS, Cancer Res 2010).

While the biochemical functions of the PTP family is known to somedegree, the therapeutic implication of the PTPRK enzyme has rarely beenexplored, particularly in relation to wound healing.

We, therefore, have surprisingly discovered that PTPRK has a role toplay in wound healing. Indeed, we have discovered that the expression ofthis protein impedes the wound healing process. Moreover, the inhibitionof PTPRK promotes wound healing.

Inhibitors of PTPRK are known. The most readily available is a salt ofstibogluconate. Sodium stibogluconate is a medicine used to treatleishmaniasis, a disease resulting from infection by one of over 20different species of the Leishmania species of parasite. SodiumStibogluconate belongs to the class of medicines known as thepentavalent antimonials. Whilst its exact paracidal effect on theLeishmania parasite is unknown it is thought that the parasite is killedby inhibition of glucose catabolism resulting in reduced ATP synthesis,thereby decreasing subsequent macromoleular synthesis and preventingreplication.

Sodium stibogluconate is sold in the United Kingdom as Pentostam™(manufactured by GlaxoSmithKline) and is currently only available foradministration by injection. Unfortunately, widespread resistance tothis medicine has limited the utility of sodium stibogluconate, and inmany parts of the world, amphotericin or miltefosine is used instead totreat leishmaniasis.

In summary, we have identified at least one molecular target fortreating wounds and in particular human wounds. More particularly, butnot exclusively, said molecular target has application in the treatmentof chronic wounds. The molecular target is PTPRK and therefore theinvention relates to a novel therapeutic comprising an inhibitor ofeither, or both, PTPRK expression or PTPRK activity. In the formerinstance, the invention involves a novel gene therapy approach and inthe latter instance a novel protein therapy approach. Accordingly, theinvention also relates to a novel therapeutic comprising an inhibitor ofeither, or both, PTPRK expression or PTPRK activity. In the formerinstance, the invention involves a novel gene therapy approach and inthe latter instance a novel protein therapy approach.

Reference herein to PTPRK, is reference to a gene or protein whoseidentity is shown in FIG. 16.

Our invention can improve the quality of a patient's life by ensuringthat new wounds do not deteriorate into a chronic state and existingchronic wounds can be treated in a way that actively promotes healing.

STATEMENTS OF INVENTION

Accordingly, in one aspect of the invention there is provided atherapeutic comprising an inhibitor of either, or both, PTPRK geneexpression or PTPRK protein activity for use in the treatment of wounds.

In the former instance, the invention involves a novel gene therapyapproach and in the latter instance a novel protein therapy approach.Thus, in one embodiment the novel therapeutic comprises an inhibitor ofPTPRK gene expression, this inhibitor can be either anti-sense DNA orRNA, siRNA, or ribozymes, either naked or in the form of plasmid andviral vectors. Those skilled in the art are aware of the aforementionedinhibitory molecules and so would be able to work the invention oncethey knew that expression of PTPRK contributed to the chronic woundphenotype. However, in another embodiment the novel therapeuticcomprises an inhibitor of PTPRK protein function, this inhibitor can beeither a PTPRK binding agent that binds, either reversibly orirreversibly, to inhibit protein function such as an antibody or aknown, or synthesized, PTPRK antagonist; or an agent that works upstreamor downstream of the PTPRK signalling mechanism to inhibit PTPRKsignalling and so negate the effects of expression of PTPRK protein inwound tissue. Those skilled in the art are aware of the aforementionedinhibitory molecules and so would be able to work the invention oncethey knew that expression of PTPRK contributed to the chronic woundphenotype.

In a preferred embodiment of the invention the therapeutic comprises aPTPRK gene inhibitor such as transgene 1 or transgene 2 or transgene 3described herein. These molecules are termed anti-PTPRK ribozyme/RNAtransgenes. Transgene 1 is produced by transcription of the PTPRK geneusing the following short oligos:

Anti-PTPRK transgene1F Ctgcagagtgagttacacagcctgatgagtccgtgagga AndAnti-PTPRK transgene1R ActagtgacaaaaactgaccaggatttgtAtttcgtcctcacggact.

Transgene 2 is produced by transcription of the PTPRK gene using thefollowing short oligos:

Anti-PTPRK transgene2F Ctgcaggatgataggaccatcgccaatctgatgagtccgtgagga andAnti-PTPRK transgene2R ActagtgatccaactaaatgccaactcgAtttcgtcctcacggact.

Transgene 3 is produced by transcription of the PTPRK gene using thefollowing short oligos:

Anti-PTPRK transgene3F Ctgcagtttgctcttttttacaattaatatctgatgagtccgtgaggaand Anti-PTPRK transgene3R ActagttcatcctccttctcctagttGtttcgtcctcacggact.

These products are antisense-hammerhead ribozyme also known asantisense-hammerhead RNA, ideally they are flanked by selectedrestriction sites such as pstI and SpeI and more ideally still they arecloned into a cloning vector such as pEF6N5His-TOPO vector (Invitrogen),

The sequence structure of transgene 1 is:

5′Ctgcagagtgagttacacagcctgatgagtccgtgaggacgaaa tacaaatcctggtcagtttttgttactagt′3

The sequence structure of transgene 2 is:

5′Ctgcaggatgataggaccatcgccaatctgatgagtccgtgaggacgaaatcgagttggcatttagttggatcactagt′3

The sequence structure of transgene 3 is:

Ctgcagtttgctcttttttacaattaatatctgatgagtccgtgaggacgaaacaactaggagaaggaggatgaactagt′3

In a preferred embodiment of the invention the therapeutic comprises acommercially available PTPRK protein inhibitor such as, withoutlimitation, Stibogluconate (GSK) or (Santa Cruz Biotechnologies Inc.,Tocris and Sigma-Aldrich).

In a further preferred embodiment of the invention the therapeuticcomprises a commercially available PTPRK protein inhibitor, such asPentostam™ (GlaxoSmithKline).

The therapeutic of the invention is for use in treating, ideally,mammalian wounds, more ideally chronic mammalian wounds, and, moreideally still, chronic human wounds. Chronic wounds that are preferablytreated using the invention are venous ulcers, diabetic ulcers, andpressure ulcers. Preferably, the wounds to be treated are non-parasitici.e. not caused—by or occupied by parasites.

An antibody for use in the invention is most ideally a monoclonalantibody or a humanised antibody.

In the above aspects and embodiments of the invention the therapeutic isformulated for topical application.

Alternatively, in the above aspects and embodiments of the invention thetherapeutic is formulated for oral application.

Alternatively again, in the above aspects and embodiments of theinvention the therapeutic is formulated for application to a dressing orimpregnation in a dressing.

The therapeutic of the invention may be administered in combination withan antibiotic or antibacterial agent. Numerous such agents are known andsuitable choices will be familiar to skilled practitioners.

In yet another aspect of the invention, there is provided apharmaceutical composition for use in treating wounds comprising atherapeutic of the invention together with a pharmaceutically acceptablecarrier.

Other active materials may also be present in the pharmaceuticalcomposition, as may be considered appropriate or advisable for the woundbeing treated. For example, the composition may also contain anemollient, or the like.

The carrier, or, if more than one be present, each of the carriers, mustbe acceptable in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipient.

The formulations include those suitable for topical (including eyedrops), oral (including buccal and sublingual), rectal, nasal or vaginaladministration and may be prepared by any methods well known in the artof pharmacy.

The composition may be prepared by bringing into association thetherapeutic of the invention and the carrier. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active agent with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product. Theinvention extends to methods for preparing a pharmaceutical compositioncomprising bringing a therapeutic of the invention in conjunction orassociation with a pharmaceutically or veterinarily acceptable carrieror vehicle.

For topical application to the skin, compounds of conventional use maybe made up into a cream, ointment, jelly, solution or suspension etc.Cream or ointment formulations that may be used for the composition areconventional formulations well known in the art, for example, asdescribed in standard text books of pharmaceutics such as the BritishPharmacopoeia.

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, sachets or tablets eachcontaining a predetermined amount of the active agent; as a powder orgranules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion; or as a bolus etc.

For compositions for oral administration (e.g. tablets and capsules),the term “acceptable carrier” includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured or inert base base andmouthwashes comprising the active agent in a suitable liquid carrier.

In a further aspect of the invention there is provided a method fortreating a mammalian wound, typically a chronic wound, which methodcomprises: administering to said wound a therapeutic that inhibitseither, or both of, PTPRK gene expression or PTPRK protein activity.

Additionally, or alternatively, the further aspect of the inventionalso, or alternatively, comprises a novel method for treating amammalian wound, typically a chronic wound, which method comprises:

administering to said wound a therapeutic that inhibits either, or bothof, PTPRK gene expression or PTPRK protein activity.

According to yet a further aspect of the invention there is provided akit for treating a wound, preferably a chronic wound, wherein said kitcomprises:

-   -   (a) at least one therapeutic as above described; and    -   (b) at least one dressing for applying to said wound.

According to a yet further aspect of the invention there is provided acombination therapeutic for treating a wound comprising an inhibitor ofPTPRK gene expression and an inhibitor of PTPRK protein activity.

According to a further aspect of the invention there is provided atherapeutic for treating a wound comprising an inhibitor of PTPRKprotein, or a homologoue thereof.

According to a further aspect of the invention there is provided use ofan inhibitor of PTPRK protein, or a homologoue thereof, in themanufacture of a medicament for treating a wound.

According to a further aspect of the invention there is provided use ofan inhibitor of PTPRK, or a homologue thereof, for treating a wound.

The term “homologue” as used herein refers to amino acid sequences whichhave a sequence at least 50% homologous to the amino acid sequence ofPTPRK and which retain the biological activity of the PTPRK sequence. Itis preferred that homologues are at least 75% homologous to the PTPRKpeptide sequence and, in increasing order of preference, at least 80%,85%, 90%, 95% or 99% homologous to the PTPRK peptide sequence.

Treatment of a wound described herein includes reference to human orveterinary use.

In the claims which follow and in the preceding description of theinvention, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprises”, or variationssuch as “comprises” or “comprising” is used in an inclusive sense i.e.to specify the presence of the stated features but not to preclude thepresence or addition of further features in various embodiments of theinvention.

Preferred features of each aspect of the invention may be as describedin connection with any of the other aspects.

Other features of the present invention will become apparent from thefollowing examples. Generally speaking, the invention extends to anynovel one, or any novel combination, of the features disclosed in thisspecification (including the accompanying claims and drawings). Thus,features, integers, characteristics, compounds or chemical moietiesdescribed in conjunction with a particular aspect, embodiment or exampleof the invention are to be understood to be applicable to any otheraspect, embodiment or example described herein, unless incompatibletherewith.

Moreover, unless stated otherwise; any feature disclosed herein may bereplaced by an alternative feature serving the same or a similarpurpose.

The present invention will now be described by way of the followingexamples with particular reference to FIGS. 1-21 wherein:

FIG. 1. Shows the secondary structure of human PTPRK mRNA;

FIG. 2. Shows HaCaT cells after lost PTPRK by way of anti-PTPRKtransgenes showed an increase in cell adhesion. Shown are traces (Toptwo panels) at 4000 Hz and 32,000 Hz and 3D modelling at 4,000 Hz and500 Hz (bottom two panels);

FIG. 3. Shows effects of knocking down PTPRK in endothelial cells on theadhesion of the cells and their response to PTPRK inhibitor,stibogluconate. Left: traces of cells, response in ECIS assays. Right:A: HECV WT, B: HECV/PTPRKrib; C: HECV wt plus stibogluconate; and D:HECV/PTPRKrib plus stibogluconate;

FIG. 4. Shows effects of knocking down PTPRK in endothelial cells oncellular migration s and their response to PTPRK inhibitor,stibogluconate. Left: traces of cells response in ECIS assays. Right: A:HECV WT, B: HECV/PTPRKrib; C: HECV wt plus stibogluconate; and D:cHECV/PTPRKrib plus stibogluconate. Cell were wounded at 6 v for 30seconds and traced immediately after wounding;

FIG. 5. Shows Traces (in triplicate) of HaCaT (WT) response tostibogluconate over an arrange of concentrations;

FIG. 6. Shows 3D modelling of HaCaT (WT) adhesion response tostibogluconate over an arrange of concentrations;

FIG. 7. Shows traces (in duplicate) of HaCaT (WT) response tostibogluconate over an arrange of concentrations. Shown are traces at100 hZ;

FIG. 8. Shows 3D modelling of HaCaT (WT) migration response tostibogluconate over an arrange of concentrations. Shown at 1000 Hz;

FIG. 9. Shows Using Rb modelling methods, a concentration dependentstimulation of cellular migration was also demonstrated. Shown are a5-hour wounding assay, with mean plus SD displayed in the graph;

FIG. 10. Shows the concentration related effect of Pentostam™(GlaxoSmithKline), a commercially available form of stibogluconate onthe migration of the cells;

FIG. 11. Shows the effect of systemic administration of sodiumstibogluconate, via the I.P. route, on the rate of wound healing.

FIG. 12 Shows effects of stibogluconate on wound healing in the db/sbmodel. The compound was given topically every other day;

FIG. 13. Shows the effect of weekly delivery of Stibogluconate on therate of wound healing;

FIG. 14. Shows the effect of twice weekly delivery of Stibogluconate onthe rate of wound healing;

FIG. 15. Shows a scatter plot of stibogluconate concentration vs size ofthe wounds after two weeks of treatment (weekly);

FIG. 16. shows the amino acid and cDNA sequence structure of PTPRK;

FIG. 17 shows the effect of removing treatment between the third andfourth week in either a weekly dosage regimen or a twice weekly dosageregimen; and Table 1. shows the primers and oligonulceotides used forthe Construction and verification of ant-human PTPRK ribozyme transgenesdescribed herein.

MATERIALS AND PROCEDURE

Cells

HaCaT, a human keratinocyte cell line was purchased from the GermanCancer Centre, HECV, a human vascular endothelial cells from Interlab,Milan, Italy, DB/DB mice from Harlan UK.

Construction of Ant-Human PTPRK Ribozyme Transgenes

The transgenes were based on the human PTPRK mRNA secondary structure(FIG. 1). Three transgenes were generated, targeting ATC and GTC sites,using respective oligos listed in Table 1. Ribozymes were generated byway of touchdown PCR, followed by verification using 2% agarose gels.The correct ribozymes were ligated into a pEF6N5His-TOPO vector(Invitrogen), followed by transformation of the ligated product to Top10E. Coli. After heat shock for 30 seconds and recover over ice for 2minutes, the bacteria was resuspended in SOC medium and allow to grow ona shaker (200 rpm) for 1 hour. The transformed bacteria were then platedover LB agar dishes which contained 100 μg/ml Ampicillin. Afterincubating the plate at 37° C. overnight, discreet colonies wereidentified and screened for the presence of the ribozyme and theorientation of the insert, by using orientation specific PCR, using T7Fprimers vs RBBMR and RBTPF primers. Correct colonies were picked, grewup in LB medium in the presence of Ampicillin. Plasmids were extracted,purified and further verified by direction specific PCR (using RBTOP vsT7F and RBBMR).

Generation of Sublines of Human Keratinocytes and Endothelial Cells withDifferential Expression of PTPRK

HaCaT and HECV cells, which were positive for PTPRK, were transfectedwith anti-PTPRK transgenes by way of electroporation (270 v). Afterselection with a selection medium (DMEM with 10 ug/ml blasticidin) for10 days, clones of selected cells were pooled and used for subsequentanalysis.

In Vivo Tolerance Test

The first tolerance test was conducted on the CD-1 athymic (CharlesRiver Laboratories). Briefly, CD-1 of 4-6 weeks old, 20 g in weight,were housed in filter topped cages. Sodium stibogluconate a known PTPRKinhibitor was injected, via the intraperitoneal route, on a daily basis.The compound was given at 100 final concentration (equivalent to ˜10mg/kg body weight) in 100 ul in volume. CD-1 were observed daily,weighed twice weekly. An additional tolerance and efficacy test wascarried out using the db/db strain.

In Vivo Efficacy Test and Wound Healing

The diabetic strain of db/db was obtained from Harlan. 4-6 weeks oldwith body weight at 20 g were used. Creation of a wound was according toa recently described method. Briefly, after being housed for a week, thedb/db were first ear-pieced using an puncher, in order to create a wound(hole) of 1 mm in diameter. The following day after wound creation, allthe db/db were weighed and the wound was photographed using a digitalcamera. Treatment was given systemically (by IP injection) or topically(by manually applying the compound in gel into the wound area). Bothtreatments were given every other day, twice weekly or weekly. Imageswere obtained weekly. The size of the wounds was determined using animage analysis software and is shown here as the area in pixels.

Effects of Knocking Down PTPRK on the Function of Cells

Three models of ECIS instrument were used: ECIS 9600 for screening andECIS1600R and ECIS Zθ for modelling. In all systems, 8W10 arrays wereused (Applied Biophysics Inc., Troy, N.Y., USA) (Giaever and Keese 1991,Kees et al 2004). Following treating the array surface with a Cysteinesolution (or array stabilization procedure for ECIS Zθ), the arrays wereincubated with complete medium for 1 hr Electric changes werecontinuously monitored for up to 24 hrs. In the 9600 system, themonitoring was at fixed 30 Hz. In the 1600R and ECIS Zθ systems, cellswere monitored at 62.5, 125, 250, 500, 1,000, 2,000, 4,000, 8,000,16,000, 32,000 and 64,000 Hz. The adhesion was analysed by theintegrated Rb modelling method.

Results

Knocking Down PTPRK from HaCaT and Endothelial Cells Resulted in anAcceleration of Cell Adhesion and Migration

It was found that after knocking down PTPRK in HaCaT cells, there was arapid increase in cell adhesion, FIG. 2. Endothelial cells, after lossof PTPRK, showed a high rate of adhesion using an ECIS assay. Likewise,HECV/WT when treated with stibogluconate, also showed a rapid adhesionto the surface of the electrode. It is interesting to observe thatHECV/PTPRKrib cells' response to stibogluconate was markedly reducedcompared with that of HECV/WT. The similar changes in cellular migrationwere seen using the electric wounding assay of the endothelial cellmodel, FIG. 3 and FIG. 4.

Human Keratinocytes Showed a Dose Dependent Response to PTPRK InhibitorStibogluconate

Using the ECIS Theta96 model tested the response of cells tostibugluconate over a range of concentrations. HaCaT cells respondedover the range of concentrations tested in that there was an increase incell adhesion between 0.16-20 uM with 20 uM showing the maximum effects,FIGS. 5 & 6. Likewise, the cells also responded to stibogluconate byincreasing their migration from concentrations as low as 160 nM to 100uM, FIGS. 7, 8 & 9.

We have also tested the concentration related effect of Pentostam™(GlaxoSmithKline), a commercially available form of stibogluconate, onthe migration of the cells, FIG. 10.

Stibogluconate was Well Tolerated In Vivo

The first tolerance test was conducted the CD-1 athymic (Charles RiverLaboratories). Briefly, CD-1 of 4-6 weeks old, 20 g in weight, werehoused in filter topped cages. Sodium stibogluconate was injected, viathe intraperitoneal route, on a daily basis. The compound was given at100 final concentration (equivalent to 10 mg/kg body weight) in 100 ulin volume. CD-1 were observed daily, weighed twice weekly. An additionaltolerance and efficacy test was carried out using the db/db strain.

Stibogluconate Accelerates Wound Healing In Vivo.

Formulation of the Compounds.

-   -   1. For systemic application, Sodium stibogluconate was dissolved        in BSS and diluted in the same for the required concentration.        The solutions were prepared that each 100 ul contained the        correct amount of compounds and was aliquatted and stored as        such at −20° C. until used. The compound was injected every        other day by the IP route.    -   2. For topical application, we used two carrier gels that are        currently used in wound care, namely Bactroban and Aquagel. From        the concentrated master stock of Sodium stibogluconate, 100 ul        of the stock solution was mixed with 2 grams of the respective        gels, followed by low speed homogenisation using a hand held        homogeniser, for 2 minutes. The newly formulated gels which        showed no signed of changes of the strength and consistency,        were stored at 4° C. until use. For use, small amount (150 ul)        of the gel was applied to the wound area and gently rubbed in        using fingers:    -   3. Sodium stibogluconate was well tolerated        -   We have delivered the compounds systemically every other            day, for a two week period in db/db. Throughout the study,            we did not observe any side effects. There was no weight            loss in any of the groups.    -   4. Sodium stibogluconate increased the rate of wound healing        without producing any side effects.        -   Sodium stibogluconate was given systemically, at 100 uM.            After one week, wounds in the treated were smaller than the            control group as shown in FIG. 1 (p=0.0927 vs control).        -   However, topical application of Sodium stibogluconate showed            no significant effect after one week, both in Bactroban and            in Aquagel (FIGS. 2 and 3).

In Vivo Test on the Dosing Effect and Exploration of the Optimal Way ofApplying the Stibogluconate

Using the same db/db mice, we further tested the possible dose responseby applying stibogluconate at 2 mg/ml. 20 mg/ml and 100 mg/ml, usingtopical applications. At the same time, we tested two treatment methods:applying the agent on a weekly basis or twice weekly basis. Wedetermined the size of the wound on a weekly basis. It was clear thatboth weekly and twice weekly application resulted in a rapid rate ofwound healing. It was also clear that the therapeutic effects ofstibogluconate is dependent on the dosage, in that the highestconcentration used, namely 100 mg/ml appear to be most effective of allthe concentrations using in the present study. Using a Two-way ANOVA(Holm-Sidak model), it was shown that in both treatment regimes, therewas a highly significant difference between the treatment group andcontrol group, p=0.013, 0.10 and 0.009, control vs 2 mg/ml, 20 mg/ml and100 mg respectively, for the twice weekly treatment, and p=0:05, 0.013,0.009 for the weekly treatment group.

Using Spearman correlation coefficient, we have found that after twoweeks treatment, the size of the wounds was significantly correlatedwith the concentration (p=0.049, r=−0.950).

Further, we have also shown that interrupting treatment, in either aweekly or twice weekly dosing regimen, prior to complete healing had asignificant effect on the healing process, resulting a noticeablereduction in wound closure (FIG. 17).

Summary

The main findings of the present study can be summarised as follows:

In wound tissue PTPRK is an important regulator of the migration ofkeratinocytes. PTPRK responds to a PTPRK inhibitor, stibogluconate, byway of increasing the adhesion and in particular migration ofkeratinocytes and also the migration of vascular endothelial cells.Moreover, Stibugluconate has a concentration dependent effect on themigration of keratinocytes. In vivo, both topical and systemicadministration of stibogluconate increased the rate of wound healing,without noticeable side effects. The effect of stibogluconate on woundhealing in vivo appears to be dose dependent. Both weekly and twiceweekly administration of stibogluconate significantly increased the rateof wound healing, although twice weekly appears to be marginally moreeffective. Interrupting the treatment regimen adversely affects thehealing process.

These findings collectively show that PTPRK is critical in controllingthe migration and healing of wounds. Thus, both in vitro and clinicaldata point to PTPRK being an important therapeutic target in wounds.

TABLE 1 Primer and oligo sequences used in the present study. Primer names Sense primers Anti-sense primers PTPRK pair aattacaattgatggggagaCcacttttccacctgaagta F11/R11 PTPRK pair aattacaattgatggggagaactgaacctgaccgtacacat ZF11/ZR11 attgtgtgacgatgaaagc PTPRK pairGcgagtcaagttatcaaacc Tgtagctgtccataagagca F12/R12 PTPRK pairgcgagtcaagttatcaaacc actgaacctgaccgtacacactcttt ZF12/ZR12 cagccatgtctagcAnti-PTPRK Ctgcagagtgagttacacagcctg Actagtgacaaaaactg accagg transgene-1atgagtccgtgagga atttgtAtttcgtcctcacggact Anti-PTPRKCtgcaggatgataggaccatcgcc Actagtgatccaactaaatgccaact transgene-2aatctgatgagtccgtgagga cgAtttcgtcctcacggact Anti-PTPRKCtgcagtttgctcttttttacaat Actagttcatcctccttctcctagt transgene-3taatatctgatgagtccgtgagga tGtttcgtcctcacggact, T7F andtaatacgactcactataggg tagaaggcacagtcgagg BGHR RBTPF andctgatgagtccgtgaggacgaa ttcgtcctcacggactcatcag RBBMR

1. A therapeutic comprising an inhibitor of one or both of PTPRK geneexpression or PTPRK protein activity or an inhibitor of a protein thatis at least 50% homologous to PTPRK for use in the treatment of wounds.2. The therapeutic according to claim 1 wherein said inhibitor is aninhibitor of PTPRK gene expression.
 3. The therapeutic according toclaim 2 wherein said inhibitor is selected from the group consisting of:anti-sense DNA or RNA, siRNA, or ribozymes, either naked or in the formof plasmid or viral vectors.
 4. The therapeutic according to claim 3wherein said inhibitor is anti-PTPRK ribozyme/RNA transgene selectedfrom the group comprising: transgene 15′Ctgcagagtgagttacacagcctgatgagtccgtgaggacgaaa tacaaatcctggtcagtttttgttactagt′3 SEQ ID NO: 7; transgene 25′Ctgcaggatgataggaccatcgccaatctgatgagtccgtgaggacgaaatcgagttggcatttagttggatcactagt′3 SEQ ID NO: 8; and transgene 3Ctgcagtttgctcttttttacaattaatatctgatgagtccgtgaggacgaaacaactaggagaaggaggatgaactagt′3 SEQ ID NO:
 9. 5. The therapeutic accordingto claim 1 wherein said inhibitor is an inhibitor of PTPRK proteinfunction.
 6. The therapeutic according to claim 5 wherein said inhibitoris selected from the group consisting of: a PTPRK binding agent thatbinds, either reversibly or irreversibly, to inhibit protein functionsuch as an antibody or a known, or synthesized, PTPRK antagonist; or anagent that works upstream or downstream of the PTPRK signallingmechanism to inhibit PTPRK function.
 7. The therapeutic according toclaim 6 wherein said inhibitor is Stibogluconate, or a salt thereof, orPentostam™ (GlaxoSmithKline).
 8. The therapeutic according to claim 1wherein the therapeutic is formulated for use in treating mammalianwounds.
 9. The therapeutic according to claim 1 wherein the therapeuticis formulated for use in treating chronic wounds comprising venousulcers, diabetic ulcers, and pressure ulcers
 10. The therapeuticaccording to claim 1 wherein the therapeutic is formulated for use intreating human wounds.
 11. The therapeutic according to claim 1 whereinthe therapeutic is formulated for topical application.
 12. Thetherapeutic according to claim 1 wherein the therapeutic is formulatedfor application to a dressing or impregnation in a dressing.
 13. Apharmaceutical composition comprising a therapeutic according to claim 1together with a pharmaceutically acceptable carrier.
 14. A method forpreparing a pharmaceutical composition according to claim 13 comprisingbringing said therapeutic in conjunction or association with apharmaceutically or veterinarily acceptable carrier or vehicle.
 15. Amethod for treating a mammalian wound which method comprises:administering to said wound a therapeutic that inhibits one or both ofPTPRK gene expression or PTPRK protein activity.
 16. A kit for treatinga wound wherein said kit comprises: (a) at least one therapeuticaccording to claim 1; and (b) at least one dressing for applying to saidwound.
 17. A combination therapeutic for treating a wound comprising aninhibitor of PTPRK gene expression and an inhibitor of PTPRK proteinactivity.
 18. A combination therapeutic for treating a wound comprising:a) either an inhibitor of PTPRK gene expression or an inhibitor of PTPRKprotein activity; and b) at least one further therapeutic. 19.(canceled)
 20. A kit for treating a wound wherein said kit comprises:(a) a composition according to claim 13; and (b) at least one dressingfor applying to said wound.